Gel electophoresis is an important tool in analytical and clinical laboratories. It takes advantage of the electrokinetic differences of ions in an electric field to accomplish separation of complex mixtures so that the components can be identified or isolated. A gel, such as agarose or polyacrylamide, is cast in a mold to produce, most commonly, an extended block of rectangular cross-section which is relatively thin. Wells are formed at one end. The block is supported and immersed in a buffer solution in apparatus having an electrode at each end. The wells are loaded with a solution containing the material under study, such as nucleic acids, and the gel is subjected to an electric field providing a high gradient along the length. The molecules migrate toward the distant electrode at different rates dependent on their mobility, which is a function of size and charge density. There are many variations of this methodology: the gels may have density gradients, either continuous or in steps; the voltage or temperature may be controlled or varied, as by controlled polarity reversal for example; and gradients may be established in pH or in concentration.
Regardless of the particular scheme employed, two basic modes of operation are used. In the usual mode, the separation is run for a finite time and then the separated molecules are read in place by some known technique which renders the pattern readable such as staining or marking with a radioactive or fluorescent material. Alternatively, the pattern is transferred in the orthogonal direction by one of various known techniques such as Southern blotting, pressure blotting, vacuum blotting, or electroblotting (transverse electric field) or the like. The separated molecules are immobilized on blotting paper or more usually, a membrane of nylon or the like where they can be identified by any of such procedures as selective dyeing, radioactive marking, fluorescence, and chemiluminescence. The procedure may employ a suitable probe to attach the marking means to the separated species.
In the other mode, often used in preparatory electrophoresis, the gel separation is run continuously (or for an extended time) and the separated species are allowed to migrate off the end of the gel into a stationary collection chamber. From time to time in a controlled manner this chamber is emptied to yield a stepwise separation of the components of the starting mixture.
In U.S. Pat. Nos. 4,631,120 and 4,631,422, Pohl teaches apparatus and method for a variant of the latter process, called direct blotting. Pohl provides one or more collecting surfaces mounted on a conveyer belt or tape and advances the belt so that it slides over the end of the gel to collect separated elemental particles and transport them with the belt away from the gel to a location where further processing such as identification may be performed. Both vertical and horizontal embodiments are disclosed. In an article by Beck and Pohl, (EMRO J, 3(12), 2905, 1984) the authors emphasize that in such a system intimate contact of the belt with the end of the gel is obtained by guiding "axles" (i.e., rollers) for the belt. The method and apparatus is described as especially amenable to automation.
A disadvantage of the apparatus of Pohl is the lack of planar stability and tension control of the collecting surface which is inherent in use of a tape or belt supported only at relatively remote lines of contact by rollers. Contact pressure is poorly controlled. Additionally contact of a roller on the collecting surface is possibly deleterious by both damage to the surface and by possible removal of some of the separated species and subsequent reprinting of the material removed onto the surface at an incorrect location.
Further, the apparatus of Pohl is not adapted to the handling and identification of a multiplicity of samples in sequence especially when the separated elements are transferred to membranes and these must be transported through a number of subsequent and different processing steps to carry out identification or the like.
U.S. Pat. No. 3,948,753 to Arlinger discloses apparatus for isotachophoretical separation, consisting of a capillary tube communicating with the sampling zone of a sampling column between electrolytes of different electrical potentials.
U.S. Pat. No. 3,705,845 to Everaerts discloses a method in counterflow isotachophoresis. A column with two electrodes holds a sample comprising ions of different mobility and to be separated. First and second electrolytes having higher and lower, respectively, mobility than the sample ions are introduced. After voltage and pressure are applied, a boundary between ion zones is detected and a control signal generated.
U.S. Pat. No. 3,047,489 to Raymond is directed to apparatus for zone electrophoresis. It discloses a preferred array of buffer medium chambers, associated electrodes, cooling plates and various buffers and wicks. U.S. Pat. No. 3,129,158 to Raymond et al., disclose a process for gel electrophoresis, including means to prepare the gel in preferred dimension, shape and position for conducting electrical current through the gel and for applying specimen mixture thereto.
U.S. Pat. No. 3,062,731 to Durrum discloses an agar-agar system, and additive suitable for electrophoretic analysis.
U.S. Pat. No. 4,622,124 is directed to a device for horizontal electroblotting of electrophoretically transferred material. A liquid tight container is provided having a support assembly horizontally disposed in a chamber defined by the container. Electrodes are located below and above the support assembly, and a bubble diverting barrier is provided to prevent uneven blotting.
U.S. Pat. No. 4,589,965 discloses a method for electroblotting whereby an electrophoretically resolved material in a gelatin sheet is transferred to a membrane with high pattern definition and resolution. The gelatin sheet is contacted with an immobilizing material, with the combination sandwiched between two plate electrodes. An electric current is applied to the electrodes of the assembly.
U.S. Pat. No. 3,674,678 discloses an apparatus for conducting electrophoretic analysis comprising a container filled with a conductive gel, two electrodes in the container, and means for effecting voltage across the gel. The container includes deep reservoirs at opposing ends and mid-section wells to accommodate samples.
It is an object of the present invention to provide electrophoretic apparatus that positions a transfer membrane relative to electrophoretic gel so that the electrophoretic transfer to the membrane is conducted in a controlled fashion. It is a further object of the present invention to provide this transfer so that the electrophoretic pattern upon visualization is remarkably clear and reproducible. A feature of the present invention is the means to provide a controlled tension on the transfer membrane as it contacts the electrophoretic gel. It is an advantage of the present invention that the apparatus disclosed herein is amenable to an automated process including a variety of processing stations. These and other objects, features and advantages of the invention will become apparent upon having reference to the following description.